Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) has emerged as a key technology for label-free bioanalysis of the spatial distribution of biomolecules, pharmaceuticals and other xenobiotics in tissue sections. Recent advances in instrumentation, sample preparation, multimodal workflows, quantification, analytical standardization and 'big data' processing have led to widespread utilization of MALDI MSI in pharmaceutical research. These developments have led to applications of the technology in drug discovery beyond drug disposition analysis, most notably in pharmacodynamic biomarker research and in toxicology.